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Journal of Biochemistry Advance Access published online on January 27, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp017
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Probing Structure of Heme A Synthase from Bacillus subtilis by Site-directed Mutagenesis

Tatsushi Mogi1,2,*

1Department of Biomedical Chemistry, Graduate School of Medicine, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033; 2ATP System Project, ERATO, JST, Nagatsuta, Midori-ku, Yokohama 226-0026, Japan

*To whom correspondence addressed. Tel: +81-3-5841-8202, Fax: +81-3-5841-3444, E-mail: tmogi{at}m.u-tokyo.ac.jp.

Received December 20, 2008; Accepted January 19, 2009


  Abstract

Biosynthesis of heme A from heme B is catalyzed by two enzymes, heme O and heme A synthase, in the membrane. Heme A synthase in Bacillus subtilis (CtaA) has eight transmembrane helices and oxidizes a methyl group on pyrrole ring D of heme O to an aldehyde. In this study, to explore structure of heme binding site(s) in heme A synthase, we overproduced the B. subtilis His6-CtaA in Escherichia coli and characterized spectroscopic properties of the purified CtaA. On the contrary to a previous report (Svensson, B., Andersson, K. K., and Hederstedt, L. (1996) Eur. J. Biochem. 238, 287-295), we found that two molecules of heme B were bound to CtaA. Further, we demonstrated that substitutions of His60 and His126 did not affect heme binding while His216 and His278 in the carboxy-halves are essential in heme binding. And we found that Ala substitutions of Cys191 and Cys197 in loop 5/6 reduced heme content to a half of the wild type level. On the basis of our findings, we proposed a helical wheel projection model of CtaA.


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